HUT71326A - Purification of kringle containing proteins, and especially t-pa - Google Patents

Abstract

Kringle containing proteins capable of binding to omega -amino acids can selectively be eluted from an ion exchange column by means of omega -amino acids.

Description

The present invention relates to a process for purifying kringle / Danish: pretzel / protein from protein solutions containing kringle-structured proteins.

Purification of proteins, especially proteins for therapeutic use, is a delicate process. The starting material to be purified is typically a biological fluid, such as plasma or serum, or a cell lysis product or culture medium, when the protein is produced by culturing a cell line capable of producing the protein. Regardless of the source, many undesirable proteins and other substances are usually present in the solution from which the protein of interest must be separated. Conventional cleaning operations consist of separating cell debris or the like by filtration or centrifugation, precipitation of the protein material, and separation of the protein material. In this way, a protein solution containing the desired protein is formed, although other proteins are contaminated. The protein solution is then typically loaded onto suitable purification columns such as affinity chromatography or ion exchange chromatography or the like.

Affinity chromatography is very suitable for laboratory scale purification, for example for research purposes. However, it is less attractive for industrial scale production, mainly due to the high cost of the column material but also due to its poor chemical stability. The column material becomes contaminated after a certain period of use and, as it cannot be regenerated, requires frequent replacement with new material, which increases the cost of production.

Thus, the affinity chromatography columns are not particularly suitable for the initial purification step when many impurities are present.

When purifying proteins on a commercial scale, it is advantageous to use an inexpensive and good chemical and physical stability charge in the initial operation. The material should also be resistant to high flow rates, for example 10 to 20 column volumes / hour. Typically, these requirements are met by the ion exchange column.

Proteins are usually liberated from the ion exchange column by changing the pH and / or ionic strength. The protein in question and the impurities then appear in different eluates, depending for example on their isoelectric point / pi /. When the pi of the protein in question and the proteins to be separated are in the same range, such elution is too non-specific, since proteins of the same pi are to some extent co-lysed from the column. Therefore, additional time-consuming and costly purification operations are required, which carry the risk of increased degradation and loss of the desired protein.

EP Ο256836Β discloses a process for purifying t-PA by separating t-PA species of different molecular weights by separating t-PA species. The mixture is contacted with hydroxyapatite and the various fractions are eluted sequentially at different pH and / or salt concentrations. Thus, this method has the disadvantages mentioned above. The same applies to the process described in EP O261941A, wherein the t-PA is purified by a cation exchange column and eluted by a salt gradient elution method. In Japanese Patent Application Laid-Open No. 63-091080, t-PA is adsorbed on an adsorption support such as a controlled porous glass (0 PG), silica gel or diatomaceous earth and eluted with a solution containing tU-amino acid. The adsorption matrices are significantly different from the ion exchange material. In adsorption chromatography, the substances to be separated are mainly bound to the matrix by surface energy and Van Der Waals forces, and the separation depends on polar and spatial factors. In ion exchange chromatography, the retention forces are predominantly electrostatic and the separation depends on the ionic nature of the components.

Problems known in the art can be solved if the protein in question is resolved from the ion exchange column with an eluent capable of selectively eluting the desired protein without substantially changing pH and ionic strength during the elution procedure. If this were possible, only the protein in question would elute, while the impurities would remain on the column.

The present invention is based on the surprising perception that certain types of amino acids, the so-called. amino acids have this desirable property. Specifically, it appeared that lys-plasminogen, a derivative of plasminogen, could be selectively eluted from the S-Sepharose column by ε-aminocaproic acid / 6-AHA / high yield.

More generally, the present invention relates to a process for purifying kringle-containing proteins that are capable of binding to ω-amino acids from a protein solution containing them. The procedure consists of the following operations:

a) applying the protein solution to an ion exchange column under conditions where the kringle-containing protein is bound to the column material;

b / selectively eluting the kringle protein with a? -amino acid without substantially altering pH and ionic strength; and c / isolating the kringle-containing protein from the eluate of step b / by further suitable purification steps.

More specifically, the present invention relates to the purification of plasminogen or a plasminogen derivative such as lys-plasminogen.

Brief description of the drawings

The invention will be described in more detail with reference to the drawings. Of these • ·· · «» * ♦ ··· »44 •» · 4 4 · 4 ·

4 4 4 4 4 4 4 ·· 4 ··

Figure 4 shows the elution profile of lys-plasminogen eluted from S-Sepharose in 0.5 M NaCl;

Figure 2 shows the elution profile of lys-plasminogen from S-Sepharose eluted with 3 mM 6AHA;

Fig. 3 shows the lys-plasminogen fraction eluted with 0.5 M NaCl and 3 mM 6-AHA in unreduced SDS-PAGE / electrophoresis / sodium dodecylsulfate pliacrylamide gel;

Figure 4 shows the elution profile of the lys-plasminogen fraction eluted from affinity column / lysine-Sepharose / dialysis with 0.5 M NaCl;

Figure 5 shows the elution profile of lys-plasminogen eluted from affinity column (lysine-Sepharose) and then dialyzed with 3 mM 6-AHA;

Fig. 6 is a non-reduced SDS-PAGE electrophoretogram of the different eluted fractions of the elution profiles of the lys-Sepharose columns of Figures 4 and 5, respectively; and Fig. 7 shows the results of a plasmin analysis of lysplasminogen further purified by the lys-Sepharose column.

Before describing the invention, it may be helpful to understand some of the definitions used below to better understand it.

plasminogen; plasminogen generally refers to human glu-plasminogen.

• ♦ • · ·· ·

Plasminogen derivatives; this definition refers to degraded or mutant forms of plasminogen, including lys plasminogen, which retains at least one of the 5 kringle domains, preferably more than 3, and most preferably all 5 kringle domains. Mutations include the replacement of one or more amino acid residues in the native molecule, or the deletion or addition of amino acid residues that modifies the properties of the melecula as desired.

<0 amino acids; the free amino group of amino acids within the scope of this definition is bound to the carbon furthest from the carboxyl group.

Kringle-containing proteins; kringle-containing proteins under one or more so-called. kringle protein such as t-PA. and plasminogen.

Selective elution; this definition refers to the desired protein free of detectable contaminating protein. Protein Solution: This definition includes a biological solution or aqueous solution that contains many proteins including the desired protein.

Biological solutions; this term refers to plasma, serum or fractions thereof or to solutions obtained by extraction of organs of human or animal origin.

It refers to the change in basically constant pH + 1 pH unit, preferably +0.5 pH unit, compared to 4 · 4 • ♦ * * * 4 · PH · buffer before adding the 6 * amino acid.

The substantially constant ionic strength means that the ionic strength varies within + 20%, preferably + 10%, within the range relative to the ionic strength of the buffer before the addition of the ω-amino acid.

Although the purification method of the present invention is used to purify kringle proteins derived from biological solutions such as plasma and serum, it is also useful for purifying intracellular protein produced from culture medium or by cell lysis. The cells that make up the kringle-containing protein may be natural cells or transformed or transfected cell lines that can express the inserted DNA region encoding the kringle-like protein.

Ion exchange columns are well known in the literature. Suitable ion exchange columns are S-Sepharose and Fractogel SO2.

Preferably, the α-amino acids contain at least 4 carbon atoms in the carbon chain between the carboxyl group and the α-amino group. Examples of suitable linear L-amino acids include 4-aminobutyric acid, 5-amino-pentanoic acid, 6-amino-hexanoic acid / ε-aminocaproic acid, 6-AHA, 7-amino-heptanoic acid, 8-amino-octanoic acid, lysine and arginine. Examples of cyclic thiamino acids are trans-4-aminomethylcyclohexanecarboxylic acid (tranexamic acid) and p-aminomethylbenzoic acid.

• ·· «4 ··« «« ο 44 * • * 44 Λ '· · • 4 · · 444 ··· 4 ·· »··» · * »t V * * 4 4 ·« «44 · 4

The method of the invention is useful for purifying kringle-containing proteins that are bound in principle to the N-amino acid and elute from the ion-exchange column without any significant change in pH and / or ionic strength. The great advantage of this is that other protein contaminants bound to the column with the protein of interest remain on the column since the pH or ionic strength is not substantially altered. The selective elution of thio-amino acids is due to their binding to the kringle-containing protein so strong that it can alter the isoelectric point of the protein or cause certain conformational changes in the molecule, leading to cleavage from the column.

An important class of proteins that can be purified by the process of the present invention is the so-called. kringle-containing proteins (see, e.g., Patthy L., Blood Goagulation and Fibrinolysis 1, 153-166 / 1990). These proteins contain one or more kringle structures which are known to bind to lysine or lysine analogs such as trans-4-aminomethylcyclohexanecarboxylic acid and ε-aminocaproic acid. Such kringle-containing proteins include t-PA and t-PA analogs with various modifications of the finger growth factor and kringle domains, and various changes around the activation site, 191,843; 201.153; 207.589; 241.209; 233.013; 240.334; 293.936; European Patent Nos. 293,934 and 292,009. Other examples include plasminogen, lys-plasminogen, apolipoprotein a, hepatocyte growth factor, FXII, prothrombin, * 4 · 4 4 44444 • 44 * 4 · 4 · 4 · 44

10 and a novel protein with putative antitumor properties, uPA / Degen S.J.F. / 1991 / 20th Lindenstrom Flame Conference, Vingsted, Denmark, Abstract 164-166. side//.

Although the primary purpose of the process of the invention is for use in an initial purification step where a large amount of impurity is present, it can also be used in a subsequent or final purification step where most of the impurity has already been removed by initial ion exchange or affinity chromatography or other suitable methods.

The eluate of the ion exchange column is preferably further purified by further suitable purification operations such as dialysis, gel filtration and affinity chromatography.

The invention will now be described in more detail below. purification of plasminogen.

Human plasminogen is a single chain glycoprotein with a molecular weight of about 90,000. It circulates in plasma at a concentration of about 2 µM and can be purified from human plasma by affinity chromatography on a Sepharose column bound to a lysine molecule. The native form of plasminogen contains NH4-terminal glutamic acid and is called glu-plasminogen '. The plasminogen molecule contains 791 amino acids with 24 disulfide bridges, 5 triple-loop kringle structures and a serine protease domain.

Proteolytic digestion of the native molecule yields many lower molecular weight forms of human plasminogen.

• ··· * · ♦♦ · 0 * * • «· 9 9 ··· • · 9 · 0« · · 0 * «·» • 909 * 0 9 · 00 ·

- 11 The nomenclature used for these molecules is based on the sequence position of the N-terminal amino acid. By exposing glu-plasminogen to plasmin for a very short period of time, 77 amino groups are cleaved from glu-plasminogen to form a molecule of plasminogen, lys-plasminogen having a molecular weight of about 83,000, which plays an important role in many properties of the fibrinolytic system.

The conversion of plasminogen to active proteinase, i.e., plasmin, is the result of so-called. plasminogen activators / PAs / catalysed. Plasmin is considered to be the primary circulating fibrinolytic enzyme in higher vertebrates, but may also play an important role in other non-vascular proteolytic events.

Plasminogen and lys-plasminogen can be used as thrombolytic agents (see Anderle K. et al., Hemostasis 18, 165-175 / 1988).

Human plasminogen can be purified from plasma in many ways. New methods of purification from human plasma are all based on affinity chromatography. An affinity matrix (lysineSepharose) is prepared by covalently linking the c (amino group) of L-lysine to Sepharose. The plasminogen is then eluted with 0.2 M β-aminocarboxylic acid, pH 7.4. The 4-aminocaproic acid is equilibrated in cold phosphate buffer.

- 12 Sephadexen is removed from plasminogen by gel filtration.

The use of plasma-purified human proteins carries a certain risk of viral infection. Plasma-derived human proteins are a major concern for HIV and hepatitis infection.

To avoid the risk, it would be advantageous to produce plasminogen by recombinant DNA technology. However, attempts to produce plasminogen in transfected cells revealed that intracellular plasminogen activation and subsequent degradation prevented the production of intact recombinant plasminogen in a reasonable yield. Ξ problem solved by co-expression of plasminogen and protease inhibitor which inhibits plasminogen activation (EP 319,944).

Thus, lys-plasminogen or glu-plasminogen in BHK cells can be produced by co-expression / co-expression of lys- or glu-plasminogen and the c? -2-plasmin inhibitor, C? Glu-plasminogen or lys-plasminogen can also be coexpressed with ζλ-1-antitrypsin / AAT in BHK cells or derivatives thereof. Coexpression with AAT ensures that urokinase produced by BHK cells is inhibited by AAT. Activation of plasminogen by urokinase and plasmin-mediated proteolytic cleavage are thus reduced and high concentrations of intact plasminogen are produced. Plasminogen can be purified from the culture medium by a two step procedure, which can be carried out by ion exchange chromatography and by the presence of V * · ··························································• · · · »« · ·· • ·· ··· ··· ··

- It consists of 13 lysine-Sepharose affinity chromatography. More specifically, the ion exchange column (S-Sepharose) is washed and then eluted with 0.5 M NaCl. The eluate was passed through an affinity chromatography column (Lys-Sepharose) and the plasminogen

Isolated with 6-AHA elution.

As mentioned, transfected BHK cells also express urokinase in addition to plasminogen. The isoelectric points of plasminogen and urokinase are 6.7 to 8.1 and 8.5, respectively. Therefore, both plasminogen and urokinase are bound to the ion exchange column, while the coexpressed plasminogen activator inhibitor appears in the void volume.

Although high yields of plasminogen can be obtained by this method, it has been found that activation of plasminogen can occur to some extent through affinity chromatography. This is presumably due to the elimination of the plasminogen activator inhibitor in the prior ion exchange purification step. When the plasminogen and plasminogen activator are co-eluted from the ion-exchange column (S-Sepharose), even a small amount of plasminogen activator (urokinase) in the eluate is capable of activating plasminogen to plasmin, and thus a small amount of plasminogen product is formed.

Surprisingly, it has been found that the use of 6-AHA in the first ion-exchange purification step can provide selective elution of lysplasminogen without the simultaneous elution of urokinase. Thus, in the eluate, plasminogen is converted to plasmin,

- Inadvertent activation of 14 urokinases can be avoided and a purer product compared to the salt gradient process can be obtained.

Experimental part

Example 1

Purification of Lys-plasminogen

Lys plasminogen is prepared according to the process described in European Patent Application 319,944.

Urinary kinase / uk / secretion of BHK cells is inhibited by 1-antitrypsin, which is co-expressed with plasminogen. The presence of the activator in the plasmin chromogenic assay supports exponential product formation. The presence of uk is detected by the fibrin overlay technique (Granelli-Piperno and Reich, J. Exp. Med. I48, 223-234 / 1978).

A sample of the culture medium is separated into two identical fractions.

The S-Sepharose ion exchange column was eluted in the first step with 6-AHA buffer or 0.5 M NaCl. Both eluates were dialyzed and purified on a lysine-Sepharose column.

• · · ι

Experiment 1

S-Sepharose, elution with NaCl / 910723. assay / Equilibration Buffer: 0.02 M NaHgPO4, 0.04 M NaCl, 0.02% Tween-80, 3 mg / L aprotinin; pH 5.0.

Wash Buffer 1 = Equilibration Buffer.

Wash buffer 2 = Equilibration buffer containing 0.15 M NaCl; pH = 5.5.

3. elution buffer = equilibration buffer without aprotinin containing 0.50 M NaCl; pH = 5.5.

Aprotinin is added to the fractions after sampling for plasmin determination.

The column volume is 5 ml, 7 ml of culture medium is applied and the flow rate is 125 ml / h.

The elution profile is shown in Figure 1.

The elution profile is quite steep. Lys plasminogen is contained in peak 2.

In Figure 1, the numbers below the baseline / 1, 2 and 3 / indicate the time of addition of the various buffers / 1. Equilibration buffer, Wash buffer 2 (0.15 & NaCl, pH 5.5), and Elution buffer 3 (0.5 M NaCl, pH 5.5). Numbers Above the Baseline In SDS-PAGE / 3. Figure 1/2 refers to the number of fractions used.

Experiment 2

S-Sepharose, elution with NaCl / # 910723, trial /

Experiment 1 is repeated, except that the column is eluted in two steps: first elution with 0.02 M NaH2PO4, 0.18 M NaCl, 0.02% Tween-80 and 3 mM

6-AHA containing elution buffer (pH 5.5).

The second elution is as described in Experiment 1.

The elution profile is shown in Figure 2. The lysplasminogen is contained in peak 2. Peak 3 corresponds to various impurities.

In Fig. 2, below the baseline, the numbers / 1, 2, 3 and 4 / show the addition of different buffers / 1. Equilibration buffer, Wash buffer 2 / 0.5 M NaCl, pH 5.5 /, Elution buffer 3 / 0.18 M NaCl, 3 mM 6-AHA, pH 5.5 /, and 4 elution buffer II / 0.5 M NaCl, pH 5.5. The numbers above the baseline / 16 to 42 refer to the number of fractions used in SDS-PAGE analysis / Figure 3 /.

Figure 3 shows a non-reduced SDS-PAGE of the elution fractions from the two experiments. Together with some aprotinin, many large molecule complexes are eluted. The numbering of the bands on the electrophoretogram is as follows:

1. Standard for determination of molecular weight

2. Plasma-derived lys-plasminogen / control /

3. Culture medium used in test 910723 «· '

4.

5.

6 · 1 ·

7.

Figure 23 Figure 23

Figure 25.

Figure 26.

Fraction Fraction Fraction Fraction / 1, Experiment /

8. The culture medium used in test 910724

9. Fraction 16 of Figure 2

10. Figure 2

11. Figure 2

12. Figure 2

13. Figure 2

14. Figure 2

15th

Fraction 22

Fraction 23

Fraction 26

Fraction 30

Fraction 42

Molecular Weight Standard / 2. experiment/

It can be seen that elution with 3 ml of 6-AHA / lane 10-13 · l / pure product free of high molecular weight complexes (lys-plasminogen) results in elution with NaCl / 4-7. band / some high molecular weight impurity product.

To examine whether the isolated lys-plasminogen contained a smaller amount of plasmin (due to urokinase activation in the eluate from the ion exchange column), further purification of lysine-Sepharose was performed.

help. This cleansing process removes aprotinin, among other things.

Both eluates were dialyzed against equilibration buffer as described below and purified by affinity chromatography on lysine-Sepharose.

Column size: 5 ml, flow rate: 50 ml / h

Equilibration buffer: 0.1 M NagHPO 4, 0.02% Tween-80, 80.3 mg / L parotinin, pH 7.4

Wash Buffer 1 = Equilibration Buffer

Wash buffer 2 = Equilibration buffer without aprotinin containing 0.15 M NaCl.

Elution is performed with a gradient of 50 M to 50 mL of 0.1 M Na 2 HPO 4 containing 0.02% Tween-80 and 0-5 mM 6-AHA.

Figure 4 shows the elution / NaCl / elution profile of the dialyzed eluate from Experiment 1.

The numbers below the baseline / 1, 2, 3 and 4 / refer to the following operations:

1. Apply Pattern

2. Wash buffer 1

3. 2. wash buffer and

4. Starting the elution gradient.

The numbers above the baseline / 8, 10, 12 show the number of fractions used in the SDSPAGE procedure / 6. figure/.

Figure 5 shows the elution profile of the dialyzed eluate from Experiment 2 (6-AHA).

The numbers below the baseline are related to the following operations

1. sample application

2. Wash buffer 1

3. Wash buffer 2

Start of gradient 4 elution.

In some cases, shoulder formation may be observed, as in Figure 4. It is known in the literature that the two glycolization forms of plasminogen have different affinities for lysineSepharose.

Figure 6, which is an SDS-PAGE image of the fractions eluted from the lysine-Sepharose ppllo, shows that lys-plasminogen is sufficiently pure considering that it has undergone only two purification operations.

Meaning of track numbers:

Molecular Weight Standard 1

2. Plasma-derived lys-plasminogen

3. the elution pattern of Figure 4/910723; probe

Without 6-AHA / 1. figure/

4 = 3

5. FIG. factions

6. FIG. factions

7. FIG. factions

8. the elution pattern of Fig. 5/910724; rehearsal • · ·

- 20 with 6-AHA, Figure 2 /

9 = 8

10th the Figure 5 7th s. factions 11th the Figure 5 8th s. factions 12th the Figure 5 9th s. factions 13th the Figure 5 10th s . factions

14. Molecular Weight Standard.

Figure 7 shows the results of plasmid analysis of lys-plasminogen purified according to the present invention, i.e., purified by S-Sepharose purification with A-A / A / and NaCl-eluted / B / purified. The assay was performed on chromogenic substrate S 2251. The assay is a standard procedure where p-NA is cleaved from S 2251 (H-D-Val-Leu-Lys-pNA) by the enzymatic action of plasmin and the resulting yellow level is evaluated photometrically at 405 nm. Prior to the assay, each sample is diluted to the same protein concentration. It is clear that plasmin is not detectable in the product eluted from S-Sepharose with 6-AHA / no sync. When eluted with NaCl, the product contains plasmin, indicating that some activation of the lysplasminogen occurred in the eluate.

Claims (7)

Claims CLAIMS 1. A method for purifying kringle-containing proteins that bind to? J-amino acids from the protein solution containing them, comprising the steps of: a) applying the kringle-containing protein solution to an ion exchange column under conditions such that the kringle protein binds to the column material; b / eluting the kringle-containing protein with a C-amino acid without substantially changing pH and ionic strength; and c) isolating the kringle protein from the eluate obtained in step b) by further suitable purification operations. 2. The process of claim 1, wherein the? -Amino acid is 4-aminobutyric acid, 5-amino-pentanoic acid, 6-amino-hexanoic acid, 7-amino-heptanoic acid, 8-aminooctanoic acid, lysine, arginine or tert-aminomethylcyclohexane-1-carboxylic acid are used. 3. The method of claim 1 or 2, wherein the kringle-containing protein is t-PA or a t-PA analog. 4. The method of claim 1 or 2, wherein the protein is plasminogen or a plasminogen derivative. 5. The method of claim 4, wherein: It is claimed that the plasminogen derivative is lys-plasminogen. Process according to any one of the preceding claims, characterized in that the amino acid is 6-aminohexanoic acid (6-AHA). Process according to any one of the preceding claims, characterized in that the eluate obtained in step b / is subjected to affinity chromatography.